Chemical properties of key metabolites determine the global distribution of lichens.

In lichen symbioses, fungal secondary metabolites provide UV protection on which lichen algae such as trebouxiophycean green algae-the most prominent group of photobionts in lichen symbioses-sensitively depend. These metabolites differ in their UV absorbance capability and solvability, and thus vary in their propensity of being leached from the lichen body in humid and warm environments, with still unknown implications for the global distribution of lichens. In this study covering more than 10,000 lichenised fungal species, we show that the occurrence of fungal-derived metabolites in combination with their UV absorbance capability and their probability of being leached in warm and humid environments are important eco-evolutionary drivers of global lichen distribution. Fungal-derived UV protection seems to represent an indirect environmental adaptation in which the lichen fungus invests to protect the trebouxiophycean photobiont from high UV radiation in warm and humid climates and, by doing this, secures its carbon source.

The Charge Distribution on a Protein Surface Determines Whether Productive or Futile Encounter Complexes Are Formed.

Protein complex formation depends strongly on electrostatic interactions. The distribution of charges on the surface of redox proteins is often optimized by evolution to guide recognition and binding. To test the degree to which the electrostatic interactions between cytochrome c peroxidase (CcP) and cytochrome c (Cc) are optimized, we produced five CcP variants, each with a different charge distribution on the surface. Monte Carlo simulations show that the addition of negative charges attracts Cc to the new patches, and the neutralization of the charges in the regular, stereospecific binding site for Cc abolishes the electrostatic interactions in that region entirely. For CcP variants with the charges in the regular binding site intact, additional negative patches slightly enhance productive complex formation, despite disrupting the optimized charge distribution. Removal of the charges in the regular binding site results in a dramatic decrease in the complex formation rate, even in the presence of highly negative patches elsewhere on the surface. We conclude that additional charge patches can result in either productive or futile encounter complexes, depending on whether negative residues are located also in the regular binding site.

Efficient Encounter Complex Formation and Electron Transfer to Cytochrome†c Peroxidase with an Additional, Distant Electrostatic Binding Site.

Electrostatic interactions can strongly increase the efficiency of protein complex formation. The charge distribution in redox proteins is often optimized to steer a redox partner to the electron transfer active binding site. To test whether the optimized distribution is more important than the strength of the electrostatic interactions, an additional negative patch was introduced on the surface of cytochrome c peroxidase, away from the stereospecific binding site, and its effect on the encounter complex as well as the rate of complex formation was determined. Monte Carlo simulations and paramagnetic relaxation enhancement NMR experiments indicate that the partner, cytochrome c, interacts with the new patch. Unexpectedly, the rate of the active complex formation was not reduced, but rather slightly increased. The findings support the idea that for efficient protein complex formation the strength of the electrostatic interaction is more critical than an optimized charge distribution.

Assessing density functional theory in real-time and real-space as a tool for studying bacteriochlorophylls and the light-harvesting complex 2.

We use real-time density functional theory on a real-space grid to calculate electronic excitations of bacteriochlorophyll chromophores of the light-harvesting complex 2 (LH2). Comparison with Gaussian basis set calculations allows us to assess the numerical trust range for computing electron dynamics in coupled chromophores with both types of techniques. Tuned range-separated hybrid calculations for one bacteriochlorophyll as well as two coupled ones are used as a reference against which we compare results from the adiabatic time-dependent local density approximation (TDLDA). The tuned range-separated hybrid calculations lead to a qualitatively correct description of the electronic excitations and couplings. They allow us to identify spurious charge-transfer excitations that are obtained with the TDLDA. When we take into account the environment that the LH2 protein complex forms for the bacteriochlorophylls, we find that it substantially shifts the energy of the spurious charge-transfer excitations, restoring a qualitatively correct electronic coupling of the dominant excitations also for TDLDA.

Breaking Benzene Aromaticity-Computational Insights into the Mechanism of the Tungsten-Containing Benzoyl-CoA Reductase.

Aromatic compounds are environmental pollutants with toxic and carcinogenic properties. Despite the stability of aromatic rings, bacteria are able to degrade the aromatic compounds into simple metabolites and use them as growth substrates under oxic or even under anoxic conditions. In anaerobic microorganisms, most monocyclic aromatic growth substrates are converted to the central intermediate benzoyl-coenzyme A, which is enzymatically reduced to cyclohexa-1,5-dienoyl-CoA. The strictly anaerobic bacterium Geobacter metallireducens uses the class II benzoyl-CoA reductase complex for this reaction. The catalytic BamB subunit of this complex harbors an active site tungsten-bis-pyranopterin cofactor with the metal being coordinated by five protein/cofactor-derived sulfur atoms and a sixth, so far unknown, ligand. Although BamB has been biochemically and structurally characterized, its mechanism still remains elusive. Here we use continuum electrostatic and QM/MM calculations to model benzoyl-CoA reduction by BamB. We aim to elucidate the identity of the sixth ligand of the active-site tungsten ion together with the interplay of the electron and proton transfer events during the aromatic ring reduction. On the basis of our calculations, we propose that benzoyl-CoA reduction is initiated by a hydrogen atom transfer from a W(IV) species with an aqua ligand, yielding W(V)-[OH-] and a substrate radical intermediate. In the next step, a proton-assisted second electron transfer takes place with a conserved active-site histidine serving as the second proton donor. Interestingly, our calculations suggest that the electron for the second reduction step is taken from the pyranopterin cofactors rather than from the tungsten ion. The resulting cationic radical, which is distributed over both pyranopterins, is stabilized by conserved anionic amino acid residues. The stepwise mechanism of the reduction shows similarities to the Birch reduction known from organic chemistry. However, the strict coupling of protons and electrons allows the reaction to proceed under milder conditions.

The dynamic complex of cytochrome c6 and cytochrome f studied with paramagnetic NMR spectroscopy.

The rapid transfer of electrons in the photosynthetic redox chain is achieved by the formation of short-lived complexes of cytochrome b6f with the electron transfer proteins plastocyanin and cytochrome c6. A balance must exist between fast intermolecular electron transfer and rapid dissociation, which requires the formation of a complex that has limited specificity. The interaction of the soluble fragment of cytochrome f and cytochrome c6 from the cyanobacterium Nostoc sp. PCC 7119 was studied using NMR spectroscopy and X-ray diffraction. The crystal structures of wild type, M58H and M58C cytochrome c6 were determined. The M58C variant is an excellent low potential mimic of the wild type protein and was used in chemical shift perturbation and paramagnetic relaxation NMR experiments to characterize the complex with cytochrome f. The interaction is highly dynamic and can be described as a pure encounter complex, with no dominant stereospecific complex. Ensemble docking calculations and Monte-Carlo simulations suggest a model in which charge-charge interactions pre-orient cytochrome c6 with its haem edge toward cytochrome f to form an ensemble of orientations with extensive contacts between the hydrophobic patches on both cytochromes, bringing the two haem groups sufficiently close to allow for rapid electron transfer. This model of complex formation allows for a gradual increase and decrease of the hydrophobic interactions during association and dissociation, thus avoiding a high transition state barrier that would slow down the dissociation process.

An ensemble of rapidly interconverting orientations in electrostatic protein-peptide complexes characterized by NMR spectroscopy.

Protein complex formation involves an encounter state in which the proteins are associated in a nonspecific manner and often stabilized by interactions between charged surface patches. Such patches are thought to bind in many different orientations with similar affinity. To obtain experimental evidence for the dynamics in encounter complexes, a model was created using the electron transfer protein plastocyanin and short charged peptides. Three plastocyanins with distinct surface charge distributions were studied. The experimental results from chemical shift perturbations, paramagnetic relaxation enhancement (PRE) NMR, and theoretical results from Monte Carlo simulations indicate the presence of multiple binding orientations that interconvert quickly and are dominated by long-range charge interactions. The PRE data also suggest the presence of highly transient orientations stabilized by short-range interactions.

Loss of electrostatic interactions causes increase of dynamics within the plastocyanin-cytochrome f complex.

Recent studies on the electron transfer complex formed by cytochrome f and plastocyanin from Nostoc revealed that both hydrophobic and electrostatic interactions play a role in the process of complex formation. To study the balance between these two types of interactions in the encounter and the final state, the complex between plastocyanin from Phormidium laminosum and cytochrome f from Nostoc sp. PCC 7119 was investigated using NMR spectroscopy and Monte Carlo docking. Cytochrome f has a highly negative charge. Phormidium plastocyanin is similar to that from Nostoc, but the net charge of the protein is negative rather than positive. NMR titrations of Zn-substituted Phormidium plastocyanin and Nostoc cytochrome f indicated that a complex with an affinity intermediate between those of the Nostoc and Phormidium complexes is formed. Plastocyanin was found in a head-on orientation, as determined using pseudocontact shifts, similar to that in the Phormidium complex, in which the hydrophobic patch represents the main site of interaction on plastocyanin. However, the interaction in the cross-complex is dependent on electrostatics, similar to that in the Nostoc complex. The negative charge of plastocyanin decreases, but not abolishes, the attraction to cytochrome f, resulting in the formation of a more diffuse encounter complex than in the Nostoc case, as could be determined using paramagnetic relaxation spectroscopy. This work illustrates the subtle interplay of electrostatic and hydrophobic interactions in the formation of transient protein complexes. The results are discussed in the context of a model for association on the basis of hydrophobic contacts in the encounter state.

Catalytic mechanism of the glycyl radical enzyme 4-hydroxyphenylacetate decarboxylase from continuum electrostatic and QC/MM calculations.

Using continuum electrostatics and QC/MM calculations, we investigate the catalytic cycle of the glycyl radical enzyme 4-hydroxyphenylacetate decarboxylase, an enzyme involved in the fermentative production of p-cresol from tyrosine in clostridia. On the basis of our calculations, we propose a five-step mechanism for the reaction. In the first step, the substrate 4-hydroxyphenylacetate is activated by an unusual concerted abstraction of an electron and a proton. Namely, Cys503 radical abstracts an electron from the substrate and Glu637 abstracts a proton. Thus in total, a hydrogen atom is abstracted from the substrate. In the second step, the carboxylic group readily splits off from the phenoxy-acetate radical anion to give carbon dioxide. This decarboxylation step is coupled to a proton transfer from Glu637 back to the phenolic hydroxyl group which leads to a p-hydroxybenzyl radical. The remaining steps of the reaction involve a rotation of the Cys503 side chain followed by a proton transfer from Glu505 to Cys503 and a hydrogen atom transfer from Cys503 to the p-hydroxybenzyl radical to form p-cresol. The calculated mechanism agrees with experimental data suggesting that both Cys503 and Glu637 are essential for the catalytic function of 4-hydroxyphenylacetate decarboxylase and that the substrate requires a hydroxyl group in para-position to the acetate moiety.

Role of hydrophobic interactions in the encounter complex formation of the plastocyanin and cytochrome f complex revealed by paramagnetic NMR spectroscopy.

Protein complex formation is thought to be at least a two-step process, in which the active complex is preceded by the formation of an encounter complex. The interactions in the encounter complex are usually dominated by electrostatic forces, whereas the active complex is also stabilized by noncovalent short-range forces. Here, the complex of cytochrome f and plastocyanin, electron-transfer proteins involved in photosynthesis, was studied using paramagnetic relaxation NMR spectroscopy. Spin labels were attached to cytochrome f, and the relaxation enhancements of plastocyanin nuclei were measured, demonstrating that a large part of the cytochrome f surface area is sampled by plastocyanin. In contrast, plastocyanin is always oriented with its hydrophobic patch toward cytochrome f. The complex was visualized using ensemble docking, showing that the encounter complex is stabilized by hydrophobic as well as electrostatic interactions. The results suggest a model of electrostatic preorientation before the proteins make contact, followed by the formation of an encounter complex that rapidly leads to electron-transfer active conformations by gradual increase of the overlap of nonpolar surface areas on cytochrome f and plastocyanin. In this model the distinction between the encounter and active complexes vanishes, at least in the case of electron-transfer complexes, which do not require a high degree of specificity.

pK(a) values and redox potentials of proteins. What do they mean?

In this article, we review a microstate model that uses protonation and redox microstates in order to understand the complex pH and redox titration of proteins and other polyelectrolytes. From this model, it becomes obvious that it is impossible to assign pK(a) values or redox potentials to individual protonatable or redox-active sites in a protein in which many of such sites interact. Instead each site is associated with many microscopic equilibrium constants that may lead to irregular or even non-monotonic titration curves of some groups. The microstate model provides a closed theoretical framework to discuss such phenomena.

A mathematical view on the decoupled sites representation.

The decoupled sites representation (DSR) is a theoretical instrument which allows to regard complex pH titration curves of biomolecules with several interacting proton binding sites as composition of isolated, non-interacting sites, each with a standard Henderson-Hasselbalch titration curve. In this work, we present the mathematical framework in which the DSR is embedded and give mathematical proofs for several statements in the periphery of the DSR. These proofs also identify exceptions. To apply the DSR to any molecule, it is necessary to extend the set of binding energies from R to a stripe within C. An important observation in this context is that even positive interaction energies (repulsion) between the binding sites will not guarantee real binding energies in the decoupled system, at least if the molecule has more than four proton binding sites. Moreover, we show that for a given overall titration curve it is not only possible to find a corresponding system with an interaction energy of zero but with any arbitrary fix interaction energy. This result also effects practical work as it shows that for any given titration curve, there is an infinite number of corresponding hypothetical molecules. Furthermore, this implies that--using a common definition of cooperative binding on the level of interaction energies--a meaningful measure of cooperativity between the binding sites cannot be defined solely on the basis of the overall titration. Consequently, all measures of cooperativity based on the overall binding curve do not measure the type of cooperativity commonly defined on the basis of interaction energies. Understanding the DSR mathematically provides the basis of transferring the DSR to biomolecules with different types of interacting ligands, such as protons and electrons, which play an important role within electron transport chains like in photosynthesis.

Virtual Model Compound Approach for Calculating Redox Potentials of [Fe2S2]-Cys4 Centers in Proteins - Structure Quality Matters.

The midpoint potential of the [Fe2S2]-Cys4-cluster in proteins is known to vary between -200 and -450 mV. This variation is caused by the different electrostatic environment of the cluster in the respective proteins. Continuum electrostatics can quantify the impact of the protein environment on the redox potential. Thus, if the redox potential of a [Fe2S2]-Cys4-cluster model compound in aqueous solution would be known, then redox potentials in various protein complexes could be calculated. However, [Fe2S2]-Cys4-cluster models are not water-soluble, and thus, their redox potential can not be measured in aqueous solution. To overcome this problem, we introduce a method that we call Virtual Model Compound Approach (VMCA) to extrapolate the model redox potential from known redox potentials of proteins. We carefully selected high-resolution structures for our analysis and divide them into a fit set, for fitting the model redox potential, and an independent test set, to check the validity of the model redox potential. However, from our analysis, we realized that the some structures can not be used as downloaded from the PDB but had to be re-refined in order to calculate reliable redox potentials. Because of the re-refinement, we were able to significantly reduce the standard deviation of our derived model redox potential for the [Fe2S2]-Cys4-cluster from 31 mV to 10 mV. As the model redox potential, we obtained -184 mV. This model redox potential can be used to analyze the redox behavior of [Fe2S2]-Cys4-clusters in larger protein complexes.

pH-dependent molecular dynamics of vesicular stomatitis virus glycoprotein G.

Vesicular stomatitis virus glycoprotein G (VSV-G) belongs to a new class of viral fusion proteins (Class III). The structure of VSV-G has been solved in two different conformations and fusion is known to be triggered by low pH. To investigate Class III fusion mechanisms, molecular dynamics simulations were performed on the VSV-G prefusion structure in two different protonation states: at physiological pH (pH 7) and low pH present in the endosome (pH 5). Domain IV containing the fusion loops, which need to interact with the target membrane, exhibits the highest mobility. Energetic analyses revealed weakened interaction between Domain IV and the protein core at pH 5, which can be attributed to two pairs of structurally neighboring conserved and differentially protonated residues in the Domain IV-core interface. Energetic calculations also demonstrated that the interaction between the subunits in the core of the trimeric VSV-G is strengthened at pH 5, mainly due to newly formed interactions between the C-terminal loop of Domain II and the N-terminus of the adjacent subunit. A pair of interacting residues in this interface that is affected by differential protonation was shown to be the main effectors of this phenomenon. The results of this study thus enhance the mechanistic understanding of the effects of protonation changes in VSV-G.

GMCT : a Monte Carlo simulation package for macromolecular receptors.

Generalized Monte Carlo titration (GMCT) is a versatile suite of computer programs for the efficient simulation of complex macromolecular receptor systems as for example proteins. The computational model of the system is based on a microstate description of the receptor and an average description of its surroundings in terms of chemical potentials. The receptor can be modeled in great detail including conformational flexibility and many binding sites with multiple different forms that can bind different ligand types. Membrane embedded systems can be modeled including electrochemical potential gradients. Overall properties of the receptor as well as properties of individual sites can be studied with a variety of different Monte Carlo (MC) simulation methods. Metropolis MC, Wang-Landau MC and efficient free energy calculation methods are included. GMCT is distributed as free open source software at www.bisb.uni-bayreuth.de under the terms of the GNU Affero General Public License.

Serine and Cysteine Peptidases: So Similar, Yet Different. How the Active-Site Electrostatics Facilitates Different Reaction Mechanisms.

The catalytic mechanisms of serine and cysteine peptidases are similar: the proton of the nucleophile (serine or cysteine) is transferred to the catalytic histidine, and the nucleophile attacks the substrate for cleavage. However, they differ in an important aspect: cysteine peptidases form a stable ion-pair intermediate in a stepwise mechanism, while serine peptidases follow a concerted mechanism. While it is known that a positive electrostatic potential at the active site of cysteine peptidases stabilizes the cysteine anion in the ion-pair state, the physical basis of the concerted mechanism of serine peptidases is poorly understood. In this work, we use continuum electrostatic analysis and quantum mechanical/molecular mechanical (QM/MM) simulations to demonstrate that a destabilization of an anionic serine by a negative electrostatic potential in combination with a compact active site geometry facilitates a concerted mechanism in serine peptidases. Moreover, we show that an anionic serine would destabilize the protein significantly compared to an anionic cysteine in cysteine peptidases, which underlines the necessity of a concerted mechanism for serine peptidases. On the basis of our calculations on an inactive serine mutant of a natural cysteine peptidase, we show that the energy barrier for the catalytic mechanism can be substantially decreased by introducing a negative electrostatic potential and by reducing the relevant distances indicating that these parameters are essential for the activity of serine peptidases. Our work demonstrates that the concerted mechanism of serine peptidases represents an evolutionary innovative way to perform catalysis without the energetically expensive need to stabilize the anionic serine. In contrast in cysteine peptidases, the anionic cysteine is energetically easily accessible and it is a very efficient nucleophile, making these peptidases mechanistically simple. However, a cysteine is highly oxygen sensitive, which is problematic in an aerobic environment. On the basis of the analysis in this work, we suggest that serine peptidases represent an oxygen-insensitive alternative to cysteine peptidases.

Enhancing the population of the encounter complex affects protein complex formation efficiency.

Optimal charge distribution is considered to be important for efficient formation of protein complexes. Electrostatic interactions guide encounter complex formation that precedes the formation of an active protein complex. However, disturbing the optimized distribution by introduction of extra charged patches on cytochrome c peroxidase does not lead to a reduction in productive encounters with its partner cytochrome c. To test whether a complex with a high population of encounter complex is more easily affected by suboptimal charge distribution, the interactions of cytochrome c mutant R13A with wild-type cytochrome c peroxidase and a variant with an additional negative patch were studied. The complex of the peroxidase and cytochrome c R13A was reported to have an encounter state population of 80%, compared to 30% for the wild-type cytochrome c. NMR analysis confirms the dynamic nature of the interaction and demonstrates that the mutant cytochrome c samples the introduced negative patch. Kinetic experiments show that productive complex formation is fivefold to sevenfold slower at moderate and high ionic strength values for cytochrome c R13A but the association rate is not affected by the additional negative patch on cytochrome c peroxidase, showing that the total charge on the protein surface can compensate for less optimal charge distribution. At low ionic strength (44 mm), the association with the mutant cytochrome c reaches the same high rates as found for wild-type cytochrome c, approaching the diffusion limit.

Structural and Biophysical Analysis of the Phytochelatin-Synthase-Like Enzyme from Nostoc sp. Shows That Its Protease Activity is Sensitive to the Redox State of the Substrate.

Phytochelatins (PCs) are nonribosomal thiol-rich oligopeptides synthetized from glutathione (GSH) in a γ-glutamylcysteinyl transpeptidation reaction catalyzed by PC synthases (PCSs). Ubiquitous in plant and present in some invertebrates, PCSs are involved in metal detoxification and homeostasis. The PCS-like enzyme from the cyanobacterium Nostoc sp. (NsPCS) is considered to be an evolutionary precursor enzyme of genuine PCSs because it shows sufficient sequence similarity for homology to the catalytic domain of the eukaryotic PCSs and shares the peptidase activity consisting in the deglycination of GSH. In this work, we investigate the catalytic mechanism of NsPCS by combining structural, spectroscopic, thermodynamic, and theoretical techniques. We report several crystal structures of NsPCS capturing different states of the catalyzed chemical reaction: (i) the structure of the wild-type enzyme (wt-NsPCS); (ii) the high-resolution structure of the γ-glutamyl-cysteine acyl-enzyme intermediate (acyl-NsPCS); and (iii) the structure of an inactive variant of NsPCS, with the catalytic cysteine mutated into serine (C70S-NsPCS). We characterize NsPCS as a relatively slow enzyme whose activity is sensitive to the redox state of the substrate. Namely, NsPCS is active with reduced glutathione (GSH), but is inhibited by oxidized glutathione (GSSG) because the cleavage product is not released from the enzyme. Our biophysical analysis led us to suggest that the biological function of NsPCS is being a part of a redox sensing system. In addition, we propose a mechanism how PCS-like enzymes may have evolved toward genuine PCS enzymes.

Investigation of the halophilic PET hydrolase PET6 from Vibrio gazogenes.

The handling of plastic waste and the associated ubiquitous occurrence of microplastic poses one of the biggest challenges of our time. Recent investigations of plastic degrading enzymes have opened new prospects for biological microplastic decomposition as well as recycling applications. For polyethylene terephthalate, in particular, several natural and engineered enzymes are known to have such promising properties. From a previous study that identified new PETase candidates by homology search, we chose the candidate PET6 from the globally distributed, halophilic organism Vibrio gazogenes for further investigation. By mapping the occurrence of Vibrios containing PET6 homologs we demonstrated their ubiquitous prevalence in the pangenome of several Vibrio strains. The biochemical characterization of PET6 showed that PET6 has a comparatively lower activity than other enzymes but also revealed a superior turnover at very high salt concentrations. The crystal structure of PET6 provides structural insights into this adaptation to saline environments. By grafting only a few beneficial mutations from other PET degrading enzymes onto PET6, we increased the activity up to three-fold, demonstrating the evolutionary potential of the enzyme. MD simulations of the variant helped rationalize the mutational effects of those mutants and elucidate the interaction of the enzyme with a PET substrate. With tremendous amounts of plastic waste in the Ocean and the prevalence of Vibrio gazogenes in marine biofilms and estuarine marshes, our findings suggest that Vibrio and the PET6 enzyme are worthy subjects to study the PET degradation in marine environments.

Structural basis for a Kolbe-type decarboxylation catalyzed by a glycyl radical enzyme.

4-Hydroxyphenylacetate decarboxylase is a [4Fe-4S] cluster containing glycyl radical enzyme proposed to use a glycyl/thiyl radical dyad to catalyze the last step of tyrosine fermentation in clostridia. The decarboxylation product p-cresol (4-methylphenol) is a virulence factor of the human pathogen Clostridium difficile . Here we describe the crystal structures at 1.75 and 1.81 Å resolution of substrate-free and substrate-bound 4-hydroxyphenylacetate decarboxylase from the related Clostridium scatologenes . The structures show a (ßγ)(4) tetramer of heterodimers composed of a catalytic ß-subunit harboring the putative glycyl/thiyl dyad and a distinct small γ-subunit with two [4Fe-4S] clusters at 40 Å distance from the active site. The γ-subunit comprises two domains displaying pseudo-2-fold symmetry that are structurally related to the [4Fe-4S] cluster-binding scaffold of high-potential iron-sulfur proteins. The N-terminal domain coordinates one cluster with one histidine and three cysteines, and the C-terminal domain coordinates the second cluster with four cysteines. Whereas the C-terminal cluster is buried in the ßγ heterodimer interface, the N-terminal cluster is not part of the interface. The previously postulated decarboxylation mechanism required the substrate's hydroxyl group in the vicinity of the active cysteine residue. In contrast to expectation, the substrate-bound state shows a direct interaction between the substrate's carboxyl group and the active site Cys503, while His536 and Glu637 at the opposite side of the active site pocket anchor the hydroxyl group. This state captures a possible catalytically competent complex and suggests a Kolbe-type decarboxylation for p-cresol formation.